1. Field of the Invention
The present invention relates to a method for carrying out immunodiagnostic tests in which enzymes or antibodies are bound to porous carrier materials. The method according to the invention is characterized by the use of macroporous polymer membranes as such porous carrier materials. The macroporous polymer membranes are prepared by preparing a homogeneous casting solution by dispersing an insoluble filler in a solution which contains at least two incompatible polymers in amounts which lead to phase separation in the solution, applying this solution to a support, and then carrying out a precipitation coagulation.
Porous carrier materials which bind biologically active proteins such as enzymes or antibodies play an important part in various technical sectors and in clinical diagnosis. In this context the immunoassay methods are particularly important. The most widely used are competitive binding assays (RIA, EIA) and sandwich assays (IRMA, ELISA), with the ELISAs becoming increasingly important. In the ELISA (Enzyme Linked Immunosorbent Assay), the antigen to be determined is bound by a carrier-bound antibody (primary antibody) and quantitatively determined by an enzymatic second antibody (secondary antibody).
2. Description of the Related Art
The basic principle of these detection methods is described in numerous publications (Angew. Chemie 97 (1985), 141-163; Chimia 29 (3) (1975), 109). The iramobilization (binding) of the primary antibody to the porous matrix surface can in principle take place in two different ways, namely by adsorption or covalent bonding, and both methods have advantages and disadvantages. Whereas the risk of denaturation of the biochemical agents is small in the case of adsorption, there is a risk of washing out or leaching out when the assay is carried out; precisely the converse applies to the covalent method. This is why the aim is to be able to use alternative methods (both adsorptive and covalent) depending on the example of use and assay method.
Detection of the immunochemical assay reaction in the case of the ELISA method frequently takes place by a chromogenic reaction which is catalyzed by an enzyme which is located on the secondary antibody. Thus, for example, secondary antibodies labelled with peroxidase (POD) are frequently employed and, in the presence of H.sub.2 O.sub.2, induce an oxidation reaction, whereupon a colour change takes place. The chromogen is impregnated into the matrix during the course of the assay procedure, and in the case of POD-catalyzed reactions the well-known water-soluble trinder system 4-aminoantipyrine/2-hydroxy-3,5-dichlorobenzenesulphonate (H.V. Bergmeyer, Methods of Enzymatic Analysis, Vol. I, 3rd. ed., Verlag Chemie) is frequently used.
Other enzyme/chromogen systems are: alkaline phosphatase/p-nitrophenyl phosphate or .beta.-galactosidase/o-nitrophenyl galactoside. Companies marketing membrane systems for immunodiagnosis are, for example, Millipore (product: IMMUBILON.RTM.) or PALL (product: IMMUNUDYNE.RTM.) in combination with brochures which describe the assay procedure. The complete assay consists of a plurality of incubation and washing steps, with the iramobilization of the primary antibody as a rule taking place by covalent bonding. Microtiter plates or dot-blot apparatuses are available for carrying out the assays, for example from Bio Rad. Critical problems in carrying out the assays are washing out and leaching out, it being possible for the substances and agents which have previously been introduced to be washed out again during a plurality of impregnation and washing steps. This is particularly critical with the chromogens employed to date which, as a rule, cannot be covalently bonded and undergo considerable extraction, because their solubility in water is often good, in the subsequent washing procedures.
This frequently leads to falsification of the results of measurement and reduction in the sensitivity.
It was therefore desirable to have available membrane matrices for immunochemical detection reactions which are particularly suitable for the ELISA method and which display the following features:
a) possibility of both adsorptive and covalent immobilization, PA1 b) possibility of covalent iramobilization in a simple and non-damaging way and PA1 c) improved immobilization of the chromogen for substantial prevention of leaching out of the reaction colours. PA1 a) dispersing insoluble fillers in a solution which contains at least two incompatible polymers in amounts which lead to phase separation in the solution, resulting in a homogeneous casting solution, and PA1 b) applying this solution to a support and carrying out a precipitation coagulation.
It has now been found, surprisingly, that macroporous membranes composed of polymer blends described in German Offenlegungsschrift 38 09 523 are outstandingly suitable for immunodiagnostic test methods.